Immunoassay element

ABSTRACT

The immunoassay element for quantitatively analyzing an antigen by determining the change in enzymatic activity of an enzyme-labelled antigen or antibody caused by an immunological reaction. The immunoassay element comprises a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of the labelling enzyme, and a reagent layer containing a fragmenting enzyme for further fragmenting the diffusible material into a lower molecular weight product. As the non-diffusible substrate, a substrate capable of reacting solely with the lebelling enzyme and incapable of reacting the fragmenting enzyme is utilized. When an endo-active glucosidase is used as the labelling enzyme, and an exo-active glucosidase is used the fragmenting enzyme in the reagent layer, the non-diffusible substrate of the substrate layer is preferred to be an endo type selectively reactive substrate, which means a substrate having a reactivity specific to endo-active glucosidase. Highly sensitive assay is realized with high accuracy and high reproducibility and good storage stability.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a dry immunoassay element inwhich a homogeneous enzyme immunoassay is utilized. More particularly,the present invention relates to an immunoassay element comprising asubstrate layer containing a non-diffusible substrate which forms adiffusible material in the presence of a labelling enzyme and a reagentlayer containing a fragmenting enzyme for further fragmenting thediffusible material into lower molecular weight product, wherein thenon-diffusible substrate is a substrate which selectively reacts withthe labelling enzyme and avoids reacting with the fragmenting enzyme.

[0003] 2. Description of the Related Art

[0004] Analyses of the constituents originated from the living body orchemicals contained in the body fluids, such as blood and urine, areuseful for diagnosing the condition of diseases or judging the course ofcuring, and thus they occupy important parts in the field of clinicaltest. The so-called enzyme immunoassay has been known in the art as onemethod for analyzing such constituents (ligands) generally present in asmall amount in the body fluids. The enzyme immunoassay may beclassified into heterogeneous systems for which B/F (Bound/Free)separation must be effected, and homogeneous system for which B/Fseparation is not necessary. The reactions in the homogeneous system arebased on the phenomenon that the enzymatic activity of the labelingenzyme is affected by some interference caused by binding of an antibodyto the antigen (ligand), and the inhibition due to antigen-antibodybinding is generally utilized. It is considered that the enzymaticactivity is suppressed by a steric hindrance caused by binding theenzyme to the substrate or a change in three-dimensional structure ofthe enzyme, when the antibody which is generally a large molecule isbound to the antigen in the enzyme-labelled antigen.

[0005] When the antigen is a high polymer, suppression of enzymaticactivity by the antigen-antibody binding reaction may be detected bylabelling the antibody with an enzyme.

[0006] Meanwhile, in the routine clinical tests in which a number oftest samples are to be handled, it is demanded that the individualsamples should be analyzed by simple operations, more desirously byautomated operation sequence.

[0007] To comply with the demand, dry analysis elements have beenproposed (see, for example, Unexamined Japanese Patent Publication Nos.53888/1974 (Corresponding to U.S. Pat. No. 3,992,158), 90859/1980(corresponding U.S. Pat. No. 4,258,001), 164356/1980 (U.S. Pat. No.4,292,272), 222769/1985 (EP 0162302A), 77356/1984 (corresponding to EP0097952A), 102388/1984 and 501866/1986 (U.S. Pat. No. 4,459,358.)

[0008] A dry analysis element has been known, in which anenzyme-labelled antibody is utilized and reacted in a homogeneous enzymeimmunological reaction (see Unexamined Japanese Patent Publication No.321360/1989 which corresponds to EP 0347839A). This known dry analysiselement comprises the following three reagent ingredients in the same ordifferent layers in the composite multi-layered structure:

[0009] (A) An antigen having a high molecular weight (a coupling productof a ligand or a derivative thereof with a high molecular weightcompound; hereinafter referred to as “polymerized antigen”);

[0010] (B) A water-insoluble high polymer substrate; and

[0011] (C) A conjugate of an antibody against the ligand and an enzymefor the substrate.

[0012] The antigen supplied by spotting onto the analysis element bindsto the antibody-enzyme conjugate through a competitive reaction with thereaction of the polymerized antigen. The complex ofantigen-antibody-enzyme reacts with the water-insoluble high polymersubstrate to form a soluble lower molecular weight product. On the otherhand, the complex of polymerized antigen and enzyme-labelled antibodyformed by the binding with the polymerized antigen cannot exhibit theenzymatic activity to the high polymer substrate. Accordingly, as thequantity of the antigen in the sample is increased, the product producedby the enzymatic reaction increases. This product is allowed to diffuseinto a detection layer where the quantity of the product is determinedby measuring the optical density of an absorption resulted by thecolored chemical group, to make it possible to analyze the antigen inthe sample quantitatively.

[0013] The immunoassay element disclosed in Japanese Patent No. 2576910(corresponding to U.S. Pat. No. 5,569,589 and EP 0451848A) is animprovement of the aforementioned immunoassay element. This immunoassayelement has a reagent layer containing a fragmenting enzyme for furtherfragmenting the decomposition product by the reaction of labellingenzyme, so that the fragmented lower molecular weight product isdetected for further sensitization of the element.

[0014] When the analyte or ligand is a macromolecular antigen, theimmunoassay element described in the specification of Japanese PatentNo. 2576913 (corresponding to U.S. patent application Ser. No.07/763,198) may be used. This prior art element has the following twocomponents either in a same layer or in different layers:

[0015] (A) Water-insoluble high polymer substrate; and

[0016] (B) Conjugate of an antibody to the macromolecular antigen and anenzyme for the substrate.

[0017] Likewise to the immunoassay element described in thespecification of Japanese Patent No. 2576910 (U.S. Pat. No. 5,569,589) areagent layer containing a fragmenting enzyme for further fragmentingthe decomposition product by the action of labelling enzyme is providedso that the fragmented product having a lower molecular weight isdetected to improve the sensitivity.

[0018] In any event, the non-diffusible substrate will not migrate intothe reagent layer. Further, it has been believed that the fragmentingenzyme will never migrate backward from the reagent layer to thesubstrate layer which overlies it. It has been heretofore customary,therefore, to deny that the substrate specificity of a non-diffusiblesubstrate is very important and therefore to select any non-diffusiblesubstrate which can react at all with both fragmenting enzyme andlabelling enzyme.

[0019] An assay element on which a sample solution has applied, however,suffers diffusion of a soluble component, if only to a slight extent,from the lower reagent layer to the upper substrate layer. The presentinventors' study of this diffusion has revealed that very small amountof the fragmenting enzyme migrates from the reagent layer to thesubstrate layer on the upstream side and reacts with the non-diffusiblesubstrate to form a low molecular weight product, and that this productconstitutes itself a noise hardly deserving to be ignored. Heretofore,such noise has had the possibility of degrading the accuracy of assay.

[0020] Further, the macromolecular substrate retained as anon-diffusible substrate in the substrate layer happens to contain asubstrate of a low polymerization degree or a substrate of an undulysmall particle diameter as an extraneous component or impurities, in aminute amount. Some of this macromolecular substrate inevitably migratesfrom the upper substrate layer to the lower reagent layer in consequenceof the advance of supply of the sample solution. The macromolecularsubstrate thus migrated in a minute amount is destined to react with thefragmenting enzyme in the reagent layer and the product of this reactionis also fated to form a cause of noise.

[0021] The present inventors, thereupon, have prepared an immunoassayelement by using as a non-diffusible substrate such a substrate asreacts solely with a labelling enzyme and avoids reacting with afragmenting enzyme and then studied this immunoassay element todetermine the quality thereof, to find that this immunoassay element issuperior to the conventional immunoassay element not only in sensitivitybut also in reproducibility and durability of aging (storage stability).

OBJECTS AND SUMMARY OF THE INVENTION

[0022] The present invention has been accomplished on the basis of thisknowledge and an object thereof is to provide an immunoassay element forenabling a high sensitive analysis of an analyte with the highestpossible accuracy and reproducibility and having high storage stability.

[0023] The object of this invention described above is accomplished byan immunoassay element for quantitatively analyzing an antigen bydetermining the change in enzymatic activity caused by any of

[0024] 1) a reaction between the antigen and an enzyme-labelledantibody;

[0025] 2) a reaction between the antigen, an antibody and anenzyme-labelled antigen; and

[0026] 3) a reaction between the antigen, an enzyme-labelled antibodyand a conjugate of the antigen with a high molecular weight compound;

[0027] wherein said element comprises a substrate layer containing anon-diffusible substrate which forms a diffusible material in thepresence of said labelling enzyme, and a reagent layer containing afragmenting enzyme for further fragmenting said diffusible material intoa lower molecular weight product:

[0028] characterized in that said non-diffusible substrate is asubstrate which reacts solely with said labelling enzyme and avoidsreacting with said fragmenting enzyme.

[0029] In a case that the subject for assay is an antibody, the objectof this invention mentioned above is accomplished by an immunoassayelement for quantitatively analyzing an antibody by determining thechange in enzymatic activity caused by a reaction between the antibodyand an enzyme-labelled antigen or a reaction between the antibody, anantigen and an enzyme-labelled antibody, wherein said element comprisesa substrate layer containing a non-diffusible substrate which forms adiffusible material in the presence of the labelling enzyme, and areagent layer containing a fragmenting enzyme for further fragmentingsaid diffusible material into a lower molecular weight product:

[0030] characterized in that said non-diffusible substrate is asubstrate which reacts solely with said labelling enzyme and avoidsreacting with said fragmenting enzyme.

[0031] To be specific, in the immunoassay element of the presentinvention, a substrate capable of reacting solely with a labellingenzyme of an antigen (or an antibody) and incapable of reacting with afragmenting enzyme contained in the reagent layer is used as anon-diffusible substrate contained in a reagent layer. As a result, thedecomposition of the non-diffusible substrate by the fragmenting enzymeis nulled and the noise is consequently abated even when the fragmentingenzyme is diffused from the reagent layer into the substrate layer onthe upstream side after the sample solution has been supplied byspotting. Even when the non-diffusible substrate happens to containextraneously such an insoluble macromolecular substrate as is notretained in the substrate layer but is migrated into the reagent layerbecause of an unduly low polymerization degree or an unduly smallparticle diameter, this insoluble macromolecular substrate will notreact with the fragmenting enzyme in the reagent layer. The use of thisnon-diffusible substrate results in improving the analyses inreproducibility and storage stability of the element.

[0032] When an endo-active glucosidase is used as the labelling enzymeof an antigen (or an antibody), and an exo-active glucosidase is usedthe fragmenting enzyme in as the reagent layer, the non-diffusiblesubstrate of the substrate layer is preferred to be an endo typeselectively reactive substrate, which means a substrate having areactivity specific to endo-active glucosidase.

[0033] Alternatively, an insoluble polysaccharide having non-reducingglucose terminal glucose positioned at the branching point of glucosechain may be used as the non-diffusible substrate having a reactivityspecific to endo-active glucosidase.

[0034] One of preferable examples of the endo type selectively reactivesubstrate is the carboxylmethylated starch which has been subjected tothe limitted decomposition with an endo-active glucosidase from thenon-reducing terminal glucose site through the carboxymethyl-modifiedglucose unit site. By using the restrictively decomposedcarboxylmethylated starch, it is made possible to heighten further thereactivity with the labelling enzyme and exalt the sensitivity of assay.

[0035] In a preferred embodiment, such a macromolecular polysaccharideas starch is used as the non-diffusible substrate, the labelling enzymefor an antigen or an antibody is to be an endo-active glucosidase whichdecomposes the macromolecular polysaccharide to a glucose oligomer, andthe fragmenting enzyme is to be an exo-active glucosidase which furtherdecomposes the oligomer into a glucose monomer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 is an illustration showing the principal layer structure ofone embodiment of the immunoassay element of the present invention;

[0037]FIG. 2 is an illustration showing another embodiment of theimmunoassay element of the present invention;

[0038]FIG. 3 is an illustration showing yet another preferred embodimentof the immunoassay element of the present invention;

[0039]FIG. 4 is a diagram showing the results of Example 4, i.e., adiagram showing the time-course changes in reflected optical densityobtained when a buffer solution containing no labelling enzyme wassupplied by spotting on the immunoassay element on a Slide 1 (workingexample) and a Slide 2 (comparative example);

[0040]FIG. 5 is a diagram showing the results of Example 4, i.e., adiagram showing the calibration curves of immunoassay element on theSlide 1 (working example) and the Slide 2 (comparative example);

[0041]FIG. 6 is a diagram showing the results of Example 6, i.e., adiagram showing the calibration curves of the immunoassay element forCRP analysis, the Slide 3 being the working example and the Slide 4being the comparative example;

[0042]FIG. 7 is a diagram showing the results of Example 12, i.e., agraphic representation demonstrating the reactivity of α-amylase to aglucoamylase-treated CM-starch (obtained in Example 9) and anon-glucoamylase-treated CM-starch (a comparative example) in a solution(dispersion) system; and

[0043]FIG. 8 is a diagram showing the results of Example 14, i.e., agraphic representation showing the time-course changes in reflectedoptical density obtained after a CRP solution of a varying concentrationwas spotted on a Slide 5 (prepared in Example 13) and a Slide 6 (acomparative example).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Layer Construction of Immunoassay Element:

[0045]FIG. 1 shows an embodiment of the immunoassay element according tothis invention. In this Figure, reference numeral 10 designates atransparent support on which laminated are reagent layer 12 and asubstrate layer 14.

[0046] The substrate layer 14 is composed of a water-permeable materialand contains a non-diffusible substrate for a labelling enzyme whichforms a conjugate with the antibody or the antigen.

[0047] The reagent layer 12 is composed of a water-permeable materialand contains a reagent composition for detecting the diffusible materialwhich has been diffused or migrated from the substrate layer. Thereagent layer 12 further contains a fragmenting enzyme for furtherfragmenting the diffusible material into a lower molecular weightproduct, so that the reagent composition detects the thus formed lowermolecular weight product.

[0048] The principal structure of the immunoassay element of theinvention, as described in the preceding paragraph, is identicalirrespective of whether the analyte is a low molecular weight antigen ora high molecular weight antigen. However, when the analyte is a lowmolecular weight antigen, the mixture is spotted on or otherwisesupplied to the substrate layer 14, the mixture containing the reactionproduct of low molecular weight antigen in the sample with theenzyme-labelled antibody and the polymerized antigen (i.e., the linkedproduct of the antigen and the high molecular weight compound). Or theanalyte antigen is mixed with the antibody and the enzyme-labelledantigen to cause a competitive reaction, and the resultant reactionmixture is spotted or supplied to the substrate layer to effect theanalysis. In any cases, the amount of the formed diffusible material isincreased as the amount or content of the ligand (low molecular weightantigen) is large or high.

[0049] On the other hand, when the analyte is a high molecular weight ormacromolecular antigen, the antigen-antibody binding reaction iseffected only between the analyte and the enzyme-labelled antibody, andthe reaction mixture is spotted on or otherwise supplied to thesubstrate layer 14. In this case, the amount of the formed diffusiblematerial is decreased as the amount of the ligand (macromolecularantigen) is large.

[0050] When the analyte is an antibody, the assay is effected byspotting or supplying to the substrate layer 14 a mixed solution whichis obtained by mixing the analyte antibody with an enzyme-labelledantigen and allowing them to react with each other. In this case, theamount of the diffusible material to be formed decreases as the amountof the analyte antibody is large or high. The assay is otherwise iseffected by spotting or supplying to the substrate layer 14 a mixedsolution which is obtained by mixing the analyte antibody with anantigen and an enzyme-labelled antibody and allowing them to undergo acompetitive reaction. In this case, the amount of the diffusiblematerial to be formed increases in proportion as the amount of theanalyte antibody to be used increases.

[0051] Analyte (Substance to be Analyzed)

[0052] The substance to be analyzed by the present invention(hereinafter referred simply as “analyte”) is an antigen or an antibody.The antigen is a ligand having an antigenic determinant and contained inthe sample.

[0053] The sample containing the analyte is not limited and many kindsof sample may be analyzed by this invention, the typical examplesincluding blood (whole blood, blood plasma, blood serum), lymph fluidand urine. It is preferred to preclude suspended particles, such asblood cells, when such particles are present. However, a sample may bedirectly spotted on the analysis element of this invention withoutprecluding such suspended particles when the analysis element has afilter layer, according to a preferred embodiment of this invention.

[0054] Any ligands, including from low molecular weigh substance to highmolecular weight substance, may be analyzed by the use of the analysiselement of this invention, as far as each ligand acts as an antigen andan antibody therefor can be provided. Incidentally, the expression “theligand has antigenicity” means that it is capable of reacting with acorresponding antibody and the ligand is only required to possess anideoptope. Even a ligand which lacks immunogenicity in itself can serveas a ligand to be analyzed contemplated by this invention so long as itis capable of producing an antibody on being immunized as a hapten.

[0055] Examples of low molecular weight antigen include medicines suchas digoxin, theophylline, phenobarbital, phenytoin, penicillin,amikacin, derivatives of these medicines (for example, complexes ofmedicines with living components, such as proteins), prostaglandin andhormones such as testosterone, progesterone and thyroxine.

[0056] Examples of macromolecular weight antigen include hormonessecreted from various endocrine glands, plasma proteins such asimmunoglobulin, albumin, ferritin, HCG (human chorionic gonadotropin)and C-reactive proteins (hereinafter referred to as CRP), viruses suchas HB antigen, bacteria, and antigens present in various organs, bloodand urine such as protein and α-phetoprotein and carcinoembryonicantigen (CEA).

[0057] When the ligand to be analyzed is an antigen having a highmolecular weight such that the ligand, on labelled with an enzyme,manifests an interfering (inhibiting) action on the enzymatic activityof the labelling enzyme, it suffices to select other low molecularsubstance having an ideoptope common to that of the ligand and use theconjugate of such substance and the labelling enzyme as anenzyme-labelled antigen, as will be described specifically herein below.The term “enzyme-labelled antigen” as used herein represents a conceptwhich embraces not only what is obtained by labelling the ligand(analyte antigen) with an enzyme but also which is obtained by labellinga substance having an ideoptope common to that of the ligand with anenzyme. Not only such compounds as ligand derivatives which areanalogues in terms of chemical structure but also such compounds asbehave similarly to ligand in terms of immune responsivity to anantibody can be labelled with an enzyme and used as an enzyme-labelledantigen.

[0058] Meanwhile, the high polymer antigens as referred to throughoutthe specification include antigens each having such a high molecularweight as exerting an interfering (suppressing) action on the enzymaticactivity of the labelling enzyme, for instance, having a molecularweight of not less than 20,000 daltons, more preferably not less thanabout 50,000 daltons. On the other hand, the low molecular weightantigens as referred to throughout the specification include antigenseach having a molecular weight low enough not to affect the enzymaticactivity of the enzyme-labelled antibody, for instance, having amolecular weight of less than 20,000 daltons. However, it should benoted here that the aforementioned specific numerical value is atentative dividing line and that the judgment on the recognition ofwhether one ligand is a low molecular weight antigen or a high molecularweight antigen should be made in consideration of the system whether thecompetition reactions between the specific analyte antigen and a certainlinked product of a high polymer compound (polymerized antigen) areutilized or not.

[0059] Polymerized Antigen

[0060] The polymerized antigen, i.e., the linked product of the ligandand a large molecule or high molecular weight compound, is bound to anantibody to suppress the activity of the enzyme which is conjugated withthe antibody for labelling the latter. The polymerized antigen is usedwhen the analyte is a low molecular weight antigen, and is not used whenthe analyte is a high molecular weight antigen.

[0061] It is preferable that the used high molecular weight compound iswater-soluble and has a molecular weight of not less than 50,000daltons. Examples of usable high molecular weight compound are proteinssuch as gelatin, hemocyanin and ferritin, and polyethylene glycol. Itsuffices that these compounds satisfy the aforementioned conditions whenbound to the ligands, and those having relatively lower molecularweights, such as bovine serum albumin, can also be used by polymerizingthem, for example, by auto-polymerization.

[0062] The method for linking the ligand to the high molecular weightcompound may be selected in considering the functional groups of theboth reactants. Utilizable functional groups include, for example,amino, carboxyl, hydroxyl, thiol, imidazole and phenyl. For example,amino groups may be linked to each other by a number of known methods,such as isocyanate method, glutaraldehyde method, difluorobenzene methodand benzoquinone methods. An amino groups may be linked to a carboxylgroup by a method in which the carboxyl group is converted tosuccinylimide ester, or by other methods including the carbodiimidemethod, Woodward reagent method and the periodic acid oxidation method(Nakane method) in which the amino group is linked with a sugar chain.When a thiol group is utilized, one of the carboxyl groups is convertedto succinylimide ester which is reacted with cysteine to introduce athiol group and then both groups are linked to each other using abifunctional linking reagent which reacts with the thiol group. Themethods in which the phenyl group is utilized include the diazotizationmethod and the alkylation method. The linking method is not limited tothe aforementioned methods, and may be selected from the methodsdescribed in “Method in Immunology and Immunochemistry”, vol. 1, (C. A.Williams, M. W. Chase, Academic Press (1967)) or “KOSO MEN'EKISOKUTEI-HO” (Enzyme Immunoassay), edited by Ishikawa, Kawai and Miyai,Igaku Shoin, 1978. The ligand may be linked to the high polymer compoundat any desired ratio. After the completion of the linking reaction, thereaction product is refined by the gel filtration or the ion exchangechromatography, and may be dried by the lyophilizing process as desired.

[0063] The ligand per se may be polymerized to obtain a polymerizedantigen. Polymerization of the ligand may be effected similar to theaforementioned linking methods. For example, the ligand may bepolymerized by using a bifunctional cross-linking agent such ascarbodiimide or glutaraldehyde.

[0064] In lieu of the ligand, the high molecular weight compound may belinked to a derivative of the ligand having immunologicalcross-reactivity to a corresponding antibody for the ligand. Meanwhile,the derivatives of the ligand include not only those which haveanalogous chemical structures but also those which exhibit analogousbehaviors in their immunological reactivities. For instance, when anantibody against theophylline as the ligand cross-reacts immunologicallywith caffeine, derivatives of caffeine may also used as materials forforming the polymerized antigen.

[0065] When the ligand or a derivative thereof has not a properfunctional group to be linked to a high molecular weight compound, anamino group, a carboxyl group or a thiol group may be introduced intothe ligand or the derivative thereof. Such a group may be introducedthrough a spacer to facilitate linking thereof to a high molecularweight compound. For example, when the ligand is theophilline, acarboxyl group may be introduced to obtain 8-propylcarboxyl-theophyllinewhich is linked to a high molecular weight compound.

[0066] Antibody

[0067] The antibody is used in the assay system in which the samplecontaining the analyte antigen is mixed with the antibody and theenzyme-labelled antigen to cause a competitive reaction. A specificantibody against the ligand which is an analyte is used for such assaysystem. When a derivative of the ligand is used for preparing theenzyme-labelled antigen, an antibody which reacts with the antigenicdeterminant common to the ligand and the derivative thereof is used. Theantibody may be a polyclonal antibody obtained by the conventionalprocess, a monoclonal antibody may be preferably used to improve thesensitivity. The antibody may be a protein fragment, such as F(ab′)₂,Fab′ or Fab.

[0068] Enzyme-Labelled Antigen

[0069] The enzyme-labelled antigen is used when a sample containing ananalyte antigen is mixed with an antibody and an enzyme-labelled antigenand they are wished to undergo a competitive reaction. It is also usedwhen the sample containing the analyte antibody is mixed with theenzyme-labelled antigen, they are allowed to undergo a binding reaction,and the amount of the antigen is determined on the basis of the changein activity of the labelling enzyme.

[0070] When the analyte is an antigen (ligand), the enzyme-labelledantigen is the linked product of the ligand (or a ligand-like substancewhich shares an ideoptope with the ligand) and an enzyme. When theligand is such a low molecular substance as a medicine, it may bedirectly bound to an enzyme. When the ligand is a substance having largemolecular weight such as a protein which, on being bound in itsunmodified form with an enzyme, interferes with the enzymatic activity,the protein may be fragmented and a fragment thereof used as a substancelabelled with an enzyme.

[0071] The protein thus fragmented and consequently allowed to acquire alowered molecular weight is only required to share an ideoptope with theintact protein (namely, the ligand).

[0072] The ligand and the enzyme may be conjugated by the same method asadopted for the linking between the antigen (ligand) and the highmolecular weight compound mentioned above.

[0073] In lieu of the ligand, the enzyme may be linked to a derivativeof the ligand having immunological cross-reactivity to a correspondingantibody for the ligand. Meanwhile, the derivatives of the ligandinclude not only those which have analogous chemical structures but alsothose which exhibit analogous behaviors in their immunologicalreactivities. For instance, when an antibody against theophylline as theligand cross-reacts immunologically with caffeine, derivatives ofcaffeine may also used as materials for forming the enzyme-labelledantigen.

[0074] When the ligand or a derivative thereof has not a properfunctional group to be linked to an enzyme, an amino group, a carboxylgroup or a thiol group may be introduced into the ligand or thederivative thereof. Such a group may be introduced through a spacer tofacilitate linking thereof to an enzyme. For example, when the ligand istheophilline, a carboxyl group may be introduced to obtain8-propylcarboxyl-theophylline which is linked to an antigen.

[0075] Enzyme-Labelled Antibody

[0076] The enzyme-labelled antibody is used when a given samplecontaining an antibody as an analyte is mixed with an antigen and anenzyme-labelled antibody and they are wished to undergo a competitivereaction. It is also used when the sample containing the antigen as ananalyte is mixed with the enzyme-labelled antibody, they are allowed toundergo a binding reaction, and the amount of the antigen is determinedon the basis of the change in activity of the labelling enzyme. When theanalyte antigen is a low molecular weight substance, the enzyme-labelledantibody is used for causing a competitive reaction of the antigencontained in the sample with a polymerized antigen and theenzyme-labelled antibody.

[0077] In the reaction system in which the competitive reaction takesplace between the analyte antibody, the antigen and the enzyme-labelledantibody, the antibody to be labelled with the enzyme is required to becapable of recognizing and reacting with an ideotope of the antigen,which should be the same as the ideotope recognized by the analyteantibody.

[0078] In the reaction system in which the competitive reaction iswished to be carried out between the analyte antigen, the polymerizedantigen, and the enzyme-labelled antibody, the antibody to be labelledwith the enzyme is required to be capable of reacting with an ideoptopewhich is common to the antigen and the polymerized antigen.

[0079] The antibody and the enzyme may be conjugated by the same methodas adopted for the linking between the antigen (ligand) and themacromolecular substance mentioned above.

[0080] Labelling Enzyme, Non-Diffusible Substrate and Fragmenting Enzyme

[0081] The enzyme bound to the antigen or the antibody as the labeldecomposes the non-diffusible high polymer substrate to produce adiffusible product, which may be fragmented or decomposed to a yet lowermolecular weight product (for example, glucose) by the action of thefragmenting enzyme.

[0082] The non-diffusible substrate is not dispersible into an aqueoussample liquid and neither diffused nor migrated into the reagent layer12 by itself.

[0083] The fragmenting enzyme s contained in the reaction layer 12 andconverts the diffusible product produced from the non-diffusiblesubstrate by the action of the labelling enzyme bound to the antigen (orthe antibody) to form a lower molecular product which can be detected.

[0084] A suitable combination of enzyme and substrate may be selected sothat an enzyme acts on the non-diffusible substrate to form a diffusiblesubstance which is further decomposed by the fragmenting enzyme toproduce a lower molecular weight product which is easily detected.

[0085] Labelling Enzyme

[0086] Examples of suitable enzyme (labelling enzyme) include hydrolaseswhich form diffusible oligomers from non-diffusible substrates composedof polymers, a specific example being glucosidase. Examples ofglucosidase includes endo-active glucosidases such as α-amylase,β-amylase, and dextranase.

[0087] It is preferred that the enzyme is not affected by any hinderingfactor present in the sample, and that a competitive enzyme of same kindis not present in the sample. However, when an enzyme which is same asthe labelling enzyme is present in the sample, an enzyme inhibitor maybe used. The enzyme inhibitor may be one which inhibits the enzyme inthe sample to a greater extent than the inhibiting activity towards thelabelling enzyme. It is most preferable that the enzyme inhibitorentirely inactivates the enzyme in the sample and does not deactivatethe labelling enzyme. However, in practical use, it suffices that theblank value is not raised at the determination step and the enzymeinhibitor may be inactivated to restore the activity of the enzyme inthe sample after the completion of determination. It also suffices ifthe enzyme inhibitor does not inhibit the enzyme in the enzyme-labelledantigen or antibody, but can inhibit the activity of free enzyme. Theenzyme inhibitor may be selected from known enzyme inhibitors so thatthe selected enzyme has the specific characteristics as aforementioned.Otherwise, an antibody against the enzyme which contained in a sample tocause a problem is prepared and used as an enzyme inhibitor.

[0088] Non-Diffusible Substrate

[0089] Examples of the substrate for said α-amylase, β-amylase ordextranase are carboxymethylated starch (also referred to as “CM-starch”hereinafter) starch, amylose and amylopectin.

[0090] It is provided, however, that the present invention uses as thenon-diffusible substrate such a substrate as reacts solely with thelabelling enzyme of the antigen (or antibody) and avoids reacting withthe fragmenting enzyme contained in the reagent layer. As a result, thedecomposition of the non-diffusible substrate by the fragmenting enzymeis nulled and the noise is consequently abated even when the fragmentingenzyme is diffused from the lower reagent layer into the upper substratelayer after the sample solution has been spotted. Even when thenon-diffusible substrate happens to contain extraneously such aninsoluble macromolecular substrate as is not retained in the substratelayer but is migrated into the reagent layer because of an unduly lowpolymerization degree or an unduly small particle diameter, thisinsoluble macromolecular substrate will not react with the fragmentingenzyme in the reagent layer.

[0091] When an α-amylase is used as the enzyme of an enzyme-labelledantibody (or antigen) and a glucoamylase or an α-glucosidase which willbe specifically described herein below is used as the fragmentingenzyme, such an insoluble polysaccharide as the starch having thenon-reducing terminal glucose thereof modified with a carboxymethylgroup may be used. Though this modified substrate can be the substrateof α-amylase, it cannot be the substrate of glucoamylase orα-glucosidase.

[0092] The modifying group of the non-reducing terminal glucose ispreferred to be a nonreactive functional group which is incapable ofaffecting the activity of a labelling enzyme or a fragmenting enzyme. Itis preferred to be a hydrophilic modifying group for the purpose ofexalting the reactivity of the enzyme in the dry assay element. Themodifying groups which answer this description include such hydrophilicgroups as a carboxylmethyl group, hydroxypropyl group, and hydroxyethylgroup. Otherwise, the terminal glucose may be esterified with phosphoricacid, sulfuric acid, or nitric acid.

[0093] Otherwise, when the non-reducing terminal glucose is bound withan adjoining glucose unit in a mode of linkage other thanα-1,4-glucoside bond (such as, for example, α-1,6 bond or α-1,3-bond)and is positioned at the point of branching of glucose chain, thepolysaccharide containing such non-reducing terminal glucose can be usedas a non-diffusible substrate contemplated by this invention. Since suchan exo-active glucosidase as glucoamylase is generally an enzyme whichdecomposes the α-1,4 bond or α-1,6 bond of the linear part of sugarchain from the non-reducing terminal into a glucose unit, it isincapable of decomposing the glucoside bond at the point of branching(mainly the α-1,6 bond or α-1,3 bond). It is, therefore, incapable ofbeing hydrolyzed even when the non-reducing terminal glucose is locatedat the position at which the non-reducing terminal glucose is branchedfrom the sugar chain.

[0094] The α-amylase is an endo-active glucosidase which hydrolyzes theα-1,4-glucoside bond of a sugar chain having not less than four glucoseunits and, therefore, is capable of being hydrolyzed within the moleculeof a substrate irrespectively of the presence or absence of amodification for the terminal glucose or the presence or absence of abranched sugar chain.

[0095] By restrictively decomposing the carboxylmethylated starch havinga carboxylmethyl group incorporated in the glucose unit halfway alongthe length of a sugar chain from the non-reducing terminal glucose sitethrough the carboxylmethyl modified glucose unit site by the exo-activeglucosidase, for example, it is made possible to convert the starch intoa substrate having the non-reducing terminal glucose thereof modifiedwith the carboxymethyl group. As concrete examples of the exo-activeglucosidase which is used herein, glucoamylase and α-glucosidase may becited.

[0096] The carboxylmethylated starch having the degree ofcarboxylmethylation thereof exalted by restrictively decomposing theterminal glucose as described above, when used in a dry assay element,has the reactivity thereof augmented with such an endo-active enzyme asα-amylase. This fact is a novel knowledge acquired by the presentinventors as will be demonstrated in the working examples to be citedherein below. Although it has not been clarified the reason why theenzymatic activity of the labelling enzyme against an insolublepolysaccharide such as starch is increased by the increase of the degreeof carboxylmethylation (the ratio of incorporation) thereof. However, itis estimated that the reactivity of the substrate to the enzyme isimproved for the following reason.

[0097] The carboxylmethyl group is a hydrophilic group. The rise of theratio of incorporation of this functional group, therefore, enables theinsoluble polysaccharide to acquire a heightened ability to hydrate andbecome readily wettable in an aqueous solvent. The reaction in a dryassay element is effected by having a sample solution of a minute amount(generally not more than 100 μL) supplied by spotting thereon and theamount of water to be supplied is extremely small. Further, since thewater (sample solution) to be supplied is spread in a pertinent layerand immediately migrated to adjoining layers as well, the interior ofthe pertinent layer which forms the field of the enzymatic reactionturns out to be an environment having an extremely limited water supply.In the layer which has such a limited water supply as mentioned above,the substrate is not allowed to swell amply and react with an enzymesufficiently. When the degree of swelling of the substrate is increased,even if only slightly, the environment under such a harsh restrictionyields to a microscopic structural change and tends to expose a reactionsite of the substrate to the enzyme. This is suspected to result inexalting the reactivity of the insoluble polysaccharide with the enzymein the dry assay element and increasing the sensitivity thereof.

[0098] In contrast, when the substrate is caused to react with an enzymein an aqueous solution, the substrate has already swelled amply and hassecured the reactivity with the enzyme. This situation may be logicallyexplained by a supposition that in the environment having an ample watersupply, the substrate is capable of swelling amply enough for thereaction and the reactivity thereof with an enzyme is not exalted anyfurther even when the degree of swelling is heightened more or less byincreasing the degree of carboxylmethylation of the substrate andconsequently enhancing the hydrophilicity thereof.

[0099] Fragmenting Enzyme

[0100] The fragmenting enzyme may be an enzyme of the same kind as ofthe labelling enzyme. In such a case, it is preferred that the labellingenzyme is an endo-active enzyme which fragments the moleculeintramolecularly to produce an oligomer, and that the fragmenting enzymeis exo-active and acts at the terminal of the molecule to produce amonomer. For instance, when the non-diffusible substrate is a polymer(e.g., starch), a fragmenting enzyme for decomposing the diffusibleoligomer (e.g., maltose) produced by the action of the labelling enzymeto a monomer (e.g., glucose) is used. Examples of the fragmenting enzymeinclude hydrolases for saccharides, specific examples being α-amylase,β-amylase, glucoamylase and α-glucosidase.

[0101] When carboxymethyl cellulose is used as the non-diffusiblesubstrate and cellulase is used as the labelling enzyme, C1 enzyme maybe used as the fragmenting enzyme. Likewise, when the combination ofgalactan and galactanase is used, β-galactosidase may be used as thefragmenting enzyme; and when the combination of RNA and ribonuclease isused, exoribonuclease may be used as the fragmenting enzyme.

[0102] The combination of the labelling enzyme, the non-diffusiblesubstrate and the fragmenting enzyme can be selected from the enzymesand substrates disclosed in prior literature (for example, “EnzymeHandbook” (compiled under supervision of Bunji Maruo and Nobuo Tamiyaand published by Asakura Shoten in 1982) and “Biochemical Handbook”(edited by Nobumasa Imura et al. and published by Maruzen Co., Ltd. in1984).

[0103] Detection System of Lower Molecular Weight Product

[0104] The lower molecular weight product produced by fragmentation inthe reagent layer by the action of the fragmenting enzyme may beoptically detected by using a known detection reagent.

[0105] Any known methods may be employed for detecting the final glucosewhich is formed by the action of the aforementioned fragmenting enzyme.Examples include:

[0106] (1) a method in which hydrogen peroxide formed by the oxidationof glucose in the presence of glucose oxidase is detected for example;

[0107] (1-1) the method wherein a Trinder reagent is used, as describedin Ann. Clin. Biochem., 6, 24 (1964) and J. Clin. Pathol., 22, 246(1969);

[0108] (1-2) the method wherein a Trinder reagent is used, as describedin Unexamined Japanese Patent Publication No. 50991/1974 (correspondingto U.S. Pat. No. 3,886,045), U.S. Pat. No. 3,992,158 and UnexaminedJapanese Patent Publication No. 164356/1980 (corresponding to U.S. Pat.No. 4,292,272);

[0109] (1-3) the method wherein a reagent containing atriaryl-substituted imidazole leuco dye is used, as described inUnexamined Japanese Patent Publication No. 26188/1978 (corresponding toU.S. Pat. No. 4,089,747) and Unexamined Japanese Patent Publication No.45557/1983 (Chemical Abstracts, 99, (1983): 209284j);

[0110] (1-4) the method wherein a reagent containing an imidazole leucodye substituted with a diarylmonoalkyl, as described in UnexaminedJapanese Patent Publication Nos. 193352/1984 (corresponding to EP0122641A) and 224677/1985 (corresponding to U.S. Pat. No. 4,665,023));

[0111] (2) a method wherein NADH produced in the presence of glucosedehydrogenase and NAD is detected; and

[0112] (3) a method wherein glucose-6-phosphate produced in the presenceof hexokinase is detected.

[0113] Among these detection methods, the most preferred is the methodwherein glucose is oxidized in the presence of glucose oxidase to formhydrogen peroxide which is detected using peroxidase and a leuco dyebecause of its high detection sensitivity.

[0114] These detection reagents may be contained in the reagent layer 12together with the fragmenting enzyme, or may be contained in anotherlayer disposed below the reagent layer 12 (for example in a secondreagent layer or a detection layer) to detect the lower molecular weightproduct produced. When a leuco dye is used, it is preferred that the dyeis dispersed in the hydrophilic binder in the solution in awater-immiscible solvent in consideration of the stability of the formeddye.

[0115] Layer Structure of the Analysis Element

[0116] The dry immunoassay element of this invention may have a layerstructure similar to those of various dry analysis element. The elementmay be of a multi-layered construction including, in addition to thesubstrate layer and the reagent layer, a support, a spreading layer, adetection layer, a light-shielding layer, an adhesive layer, awater-absorbing layer, an undercoating layer and so on. Examples of suchanalysis elements are disclosed in the specifications of UnexaminedJapanese Patent Publication Nos. 53888/1974 (corresponding to U.S. Pat.No. 3,992,158), 40191/1976 (corresponding to U.S. Pat. No. 4,042,353),164356/1980 (corresponding to U.S. Pat. No. 4,292,272) and 4959/1986(corresponding to European Patent No. 0166365A).

[0117] When a light-transmitting and water-impermeable support is used,the dry immunoassay element having the following construction may beused, although the present invention is not limited to the followingconstructions.

[0118] (1) A reagent layer disposed on a support, and a substrate layersuperposed on the reagent layer;

[0119] (2) A reagent layer disposed on a support, an adhesive layersuperposed on the reagent layer and a substrate layer superposed on theadhesive layer in this order;

[0120] (3) A support, and a detection layer, a reagent layer and asubstrate layer superposed in this order;

[0121] (4) A support, and a reagent layer, a light-shielding layer, anda substrate layer superposed in this order;

[0122] (5) A support, and a detection layer, a reagent layer,light-shielding layer and a substrate layer superposed in this order;

[0123] (6) A support, and a detection layer, a light-reflecting layer, areagent layer and a substrate layer superposed in this order;

[0124] (7) A support, and a second reagent layer, a light-reflectinglayer, a first reagent layer and a substrate layer superposed in thisorder; and

[0125] (8) A support, and a detection layer, a second reagent layer, alight-reflecting layer, a first reagent layer and a substrate layersuperposed in this order.

[0126] In the constructions (1) to (6), the reagent layer may becomposed of plural layers. The reagent layer may be an immunologicalreaction layer which contains a component capable of taking part in aimmunological reaction as will be described hereinafter.

[0127] A water-absorbing layer may be disposed between the support andthe reagent or detection layer. Filtering layers may be interposedbetween the adjacent layers. A spreading layer may be disposed on thesubstrate layer, or the substrate layer may serve also as a spreadinglayer.

[0128] Substrate Layer

[0129] The substrate layer 14 is composed of a water-permeable layer andcontains a non-diffusible substrate which is a substrate for the enzymelabelling the antibody.

[0130] In order to ensure water-permeability of the substrate layer, itis preferable that the substrate layer is composed of a porous medium ora layer composed of a hydrophilic polymer binder.

[0131] The porous layer may be fibrous or non-fibrous. As the fibrousmaterial, filter paper, non-woven cloth, woven cloth (e.g. plain wovencloth), knitted cloth (e.g. tricot knitted cloth) or filter paper madeof glass fibers may be used. Examples of the non-fibrous materialinclude a membrane filter composed of cellulose acetate described inUnexamined Japanese Patent Publication No. 53888/1974 (corresponding toU.S. Pat. No.3,992,258), and a particulate structure layer containinginter connected voids and composed of inorganic or organic fineparticles as disclosed in Unexamined Japanese Patent Publication Nos.53888/1974 (corresponding to U.S. Pat. No. 3,992,258), 90859/1980(corresponding to U.S. Pat. No. 4,258,001) and 70163/1983 (correspondingto U.S. Pat. No. 4,486,537). A laminated structure made of partiallybonded multiple porous layers may also be preferably used, examples ofsuch structure being disclosed in Unexamined Japanese Patent PublicationNos. 4549/1986 (corresponding to EP 0166265A), 116258/1987 (ChemicalAbstracts, 108, (1988): 3041y), 138756/1987 (EP 0226465A), 138757/1987(EP 0226465A) and 138758/1987 (EP 0226465A).

[0132] The porous layer may be a spreading layer having a so-calledmetering function to spread a liquid over an area substantially inproportion to the volume of the liquid fed thereto. Preferable materialsfor the spreading layer are woven and knitted fabrics. The woven fabricsor like may be subjected to the glow discharge treatment as described inUnexamined Japanese Patent Publication No. 66359/1982 (corresponding toGB 2,087,974A and U.S. Pat. No. 4,783,315). In order to adjust the areaor rate for spreading, the spreading layer may contain a hydrophilicpolymer or a surfactant as described in Unexamined Japanese PatentPublication Nos. 222770-1985 (corresponding to EP 0162301A), 219397/1988(corresponding to DE 37 17 913 A), 112999/1988 (corresponding to DE 3717 913A) and 182652/1987 (corresponding to DE 37 17 913A).

[0133] One convenient method is a method wherein the substrate isimpregnated into or coated on a porous membrane made of, for example,paper, cloth or a high polymer, and then the composite is applied onanother water-permeable layer, for example, a reagent layer superposedon the support by a method as described in unexamined Japanese PatentPublication No. 164356/1980 (corresponding to U.S. Pat. No. 4,292,272).A further method comprises the steps of bonding a porous layer onanother water-permeable layer (for example a reagent layer) by a methodas described above, and coating a composition containing the substrateon the porous layer. Any known methods may be employed for theimpregnation or coating on the porous layer. Coating may be effected byselecting a suitable method, for example, dip coating, doctor coating,hopper coating and curtain coating.

[0134] Although the thickness of the substrate layer made by any of theaforementioned methods is not limited, the thickness may range within 1μm to 50 μm and preferably, from 2 μm to 30 mm, when the layer isprovided as a coating layer. When it is provided by another method, forexample by piling of a laminate, the thickness thereof may be variedwithin a wide range of from several tens of μm to several hundreds ofμm.

[0135] The substrate layer may be a water-permeable layer composed of ahydrophilic polymer binder, such as, gelatin and derivatives thereof(e.g. phthalated gelatin), derivatives of cellulose (e.g. hydroxyethylcellulose), agarose, sodium alginate, acrylamide copolymers,methacrylamide copolymers, copolymers of acryl amides or methacrylamideswith various vinyl monomers, polyhydroxyethyl methacrylate, polyvinylalcohol, polyvinyl pyrrolidone, sodium polyacrylate, and copolymers ofacrylic acid with various vinyl monomers.

[0136] The substrate layer composed of a hydrophilic polymer binder maybe provided by coating and aqueous solution or dispersion of thesubstrate, an additional other reagent composition and a hydrophilicpolymer binder on another layer, such as a support or a detection layer,and then drying the coated solution or dispersion, as disclosed in thespecifications of Japanese Patent Publication No. 21677/1988(corresponding to U.S. Pat. No. 3,992,158), Unexamined Japanese PatentPublication Nos. 164356/1980 (corresponding to U.S. Pat. No. 4,292,272),101398/1979 (corresponding to U.S. Pat. No. 4,132,528), and 292063/1986(Chemical Abstracts, 106, (1987): 210567y). The thickness of the driedsubstrate layer containing a hydrophilic polymer as the binder may rangefrom about 2 μm to about 50 μm, and preferably, from about 4 μm to about30 μm, and the coverage thereof may range from about 2 g/m² to about 50g/m², and preferably, from about 4 g/m² to about 30 g/m².

[0137] To improve the characteristics, such as, coating characteristics,diffusibility of the diffusible material, reactivity and storagestability, the substrate layer may include, in addition to thenon-diffusible substrate, various organic or inorganic additives, forexample, enzyme activators, coenzymes, surfactants, pH buffer reagents,fine particles, antioxidants, etc. Examples of buffer system, which maybe contained in the substrate layer, include pH buffer reagents asdescribed in “KAGAKU BINRAN, KISOHEN” edited by Japanese ChemicalSociety (MARUZEN, Tokyo, 1966), pp1312-1320; R. M. C. Dawson et al.,“Data for Biological Research”, 2^(nd) Edition (Oxford at the ClarendonPress, 1969), pp476-508; “Biochemistry”, 5, pp467-477 (1966); and“Analytical Biochemistry”, 104, pp300-310 (1980). Specific examples ofusable buffers are buffer reagents containing tris (hydroxymethyl)aminomethane (Tris), buffer reagents containing phosphates, buffersolutions containing borates, buffer reagents containing citric acid orcitrates, buffer reagents containing glycine, buffer solutionscontaining Bicine, and buffer reagents containing HEPES.

[0138] Reagent Layer

[0139] The reagent layer 12 contains a reagent composition for detectingthe diffusible material which has diffused and migrated from thesubstrate layer 14. As desired, a fragmenting enzyme may be contained inthe detection reagent composition and a detection reagent compositionfor detecting the lower molecular weight product formed by the action ofthe fragmenting enzyme may also be contained.

[0140] The reagent layer 12 is composed of a water-permeable layer whichis preferably a continuous layer made of a hydrophilic polymer binder,similar to the water-permeable layers as described in the description othe substrate layer. The used hydrophilic polymer binder may bedetermined in consideration of the diffusible product formed in thesubstrate layer and the coloring reagent contained in the reagent layer.

[0141] Support

[0142] The support 10 may be light-nontransmitting (opaque),light-semi-transmitting (translucent) or light-transmitting(transparent), and it is generally preferable that the support islight-transmitting and water-impermeable. Preferable materials for thelight-transmitting and water-impermeable support are polyethyleneterephthalate and polystyrene. In general, an undercoating is providedor the support is subjected to hydrophilization treatment in order tofirmly adhere the hydrophilic layer.

[0143] Immunological Reaction Layer

[0144] The substrate layer 14 shown in FIG. 1 may containenzyme-labelled or-unlabelled immunological binding partner (antigen orantibody), in addition to the diffusible substrate, to form animmunological reaction layer in which an immunological reaction takesplace.

[0145] For example, the substrate layer is made to contain therein

[0146] (1) an enzyme-labelled antibody when the amount of an antigensubjected to the assay is determined by causing the antigen to reactwith the enzyme-labelled antibody;

[0147] (2) an antibody and an enzyme-labelled antigen when the amount ofan antigen subjected to the assay is determined by causing the antigento react with an antibody and the enzyme-labelled antigen;

[0148] (3) a polymerized antigen and an enzyme-labelled antibody whenthe amount of an antigen subjected to the assay is determined by causingthe antigen to react with the polymerized antigen and theenzyme-labelled antibody;

[0149] (4) an enzyme-labelled antigen when the amount of an antibodysubjected to the assay is determined by causing the antibody to reactwith the enzyme-labelled antigen; or

[0150] (5) an antigen and an enzyme-labelled antibody when the amount ofan antibody subjected to the assay is determined by causing the antibodyto react with the antigen and the enzyme marker antibody.

[0151] With such constructions, the substrate layer functions as animmunological reaction layer for allowing an immunological reaction toproceed additionally therein. In this case, a homogeneous enzymeimmunological reaction takes place in the element only by spotting asample solution on the element.

[0152] Alternatively, either of a given pair of reactants may becontained in the substrate layer and the remainder in a water-permeatinglayer superposed on the substrate layer. Otherwise, a water-permeatinglayer formed of one layer or a plurality of layers may be formed on thesubstrate layer and these layers may be allowed to contain immunologicalbinding partners necessary for an immunological reaction. The stratalconstruction of these immunological reaction layers can be arbitrarilydecided, depending on the mode of the immunological reaction wished tobe effected.

[0153] In the case of the reaction of (2) mentioned above, for example,the antibody and the enzyme-labelled antigen may be severally containedin a plurality of layers other than the substrate layer. For example,the immunoassay element may comprise a water-permeable layer 16containing an antigen and superposed on the substrate layer 14 andfurther comprise thereon a water-permeable layer 18 containing anenzyme-labelled antigen as illustrated in FIG. 2. In this case, theantigen (ligand) in the sample diffuses and permeates through the layer16 together with the enzyme-labelled antigen of the layer 18. In thelayer 16, the antigen and the enzyme-labelled antigen are respectivelybound with the antibody and further allowed to migrate into thesubstrate layer 14.

[0154] Conversely, the immunoassay element may comprise thewater-permeable layer 16 containing the enzyme-labelled antigen; and thewater-permeable layer 18 superposed on the layer 18 and containing theantibody. In this case, the antigen (ligand) in the sample binds withthe antibody contained in layer 18, and then migrates into the layer 16.In the layer 16, the antibody which has escaped being bound with theantigen binds with the enzyme-labelled antigen, and further migratesinto the substrate layer 14.

[0155] Optionally, one layer other than the substrate layer may containthe antibody and the enzyme-labelled antigen together in a substantiallydry state or in the substantial absence of water. For example, animmunoassay element may be constructed by forming on the substrate layer14 a water-permeable layer 20 containing the antibody and theenzyme-labelled antigen together in a substantially dry state or in thesubstantial absense of water as illustrated in FIG. 3. In this case,when the sample using water as the solvent therefor is supplied on thelayer 20, the ligand (antigen) in the sample and the enzyme-labelledantigen are bound respectively with the antibody in the wateroriginating from the sample in the layer 20, and then migrates into thesubstrate layer 14. In order to contain an antibody and anenzyme-labelled ligand together in a separate layer in the substantiallydry state or in a substantial absence of water, either or both of theantibody and the enzyme-labelled antigen may be dissolved or dispersedin a non-aqueous solvent such as an alcohol (for example, ethanol) andthen the resultant solution or dispersion is impregnated in thewater-permeable layer.

[0156] When the provision of an immunoreaction layer is omitted, theassay element of this invention can be used also for assaying an enzymecapable of decomposing the non-diffusible substrate contained in thesubstrate layer 14. When carboxylmethylated starch, starch, amylose oramylopectin is used as the non-diffusible substrate, for example, theassay element of this invention can be used for the assay of anendo-active glucosidase such as α-amylase or β-amylase.

[0157] Process for Preparing the Immunoassay Element

[0158] The dry immunoassay element of the invention may be prepared byany of the known processes described in the specifications ofaforequoted patents.

[0159] The analysis element of the invention may be cut into a squarepiece having sides each ranging from about 15 mm to about 30 mm or adisk having a substantially the same area. It is preferred, in view ofthe preparation, packaging, shipping, storage and measuring operations,that the element be contained in a slide frame as disclosed, forexample, in Japanese Patent Publication No. 28331/1982 (corresponding toU.S. Pat. No. 4,169,751), Unexamined Japanese Utility Model PublicationNo. 142454/1981 (corresponding to U.S. Pat. No. 4,387,990), UnexaminedJapanese Patent Publication No. 63452/1982, Unexamined Japanese UtilityModel Publication No. 32350/1983 and Unexamined Japanese PatentPublication No. 501144/1983 (corresponding to International PublicationWO 83/00391) for use as a slide for chemical analysis. For theconvenience in some uses, it may be formed in a tape shape which iscontained in a cassette or magazine, or a small piece thereof may beapplied on or contained ina card having an opening.

[0160] Analyzing Method Using the Immunoassay Element

[0161] The analysis element of the invention may be used for thequantitative analysis of an analyte ligand (or an analyte antibody) in asample liquid by using it through the operations described in thespecification of the aforequoted patents.

[0162] For example, about 5 μL to about 30 μL, preferably 8 μL to 15 μLof an aqueous sample liquid, such as, serum, plasma or urine, is spottedor otherwise fed on the substrate layer 14. The analysis element spottedwith the sample liquid is then incubated at a constant temperature offrom about 20° C. to about 45° C., preferably at a constant temperatureof from about 30° C. to about 40° C., for 1 to 10 minutes. Thereflection optical density of the color or the change in color in theelement may be measured from the light-transmitting support side, andthe quantity of the ligand (or antibody) contained in the sample can bedetermined using a preliminarily prepared calibration curve based on theprinciple of colorimetry. The volume of the spotted liquid sample andthe time and temperature for incubation are maintained constant toimprove the accuracy in quantitative analysis.

[0163] The measuring operation may be carried out while using thechemical analysis apparatus described in Unexamined Japanese PatentPublication Nos. 125543/1985, 220862/1965, 294367/1986 and 161867/1983(the last-mentioned Publication corresponding to U.S. Pat. No.4,424,191) to realize a quantitative analysis at a high accuracy byextremely easy operations. Meantime, a semi-quantitative analysis may beconducted by judging the degree of coloring by naked eye if such visualjudgment is adequate for the object or required accuracy.

[0164] When the analysis element has no immunological reaction layer,namely when the analysis element does not contain therein aimmunological binding partner necessary for the immunological reactionwith an antigen or antibody to be analyzed, a necessary immunologicalreaction can be carried out in a proper reaction mixture other than theassay element, and then the resultant reaction mixture is spotted on theelement. Thus the analyte can be analyzed as a change of the enzymaticactivity of the labelling enzyme. For assaying an antigen, for example,an aqueous sample solution is mixed with a solution containing anantibody and an enzyme-labelled ligand to complete the binding reaction,and then spotted on the substrate layer.

EXAMPLE 1

[0165] Preparation of Endo Type Specific Reactive Substrate;

[0166] 10 Gram of carboxylmethylated starch made by Edward MendelCompany Inc. and sold under a trademark designation of “Exprotab” wasdispersed in 1 liter of 0.1 M borato buffer solution (pH 10), and thenstirred for one hour. The resultant dispersion was adjusted withhydrochloric acid and phosphoric acid to pH 5.5, and then 1000 U ofglucoamylase (made by Toyobo K.K.) was added to react with thecarboxylmethylated starch at 37° C. for 16 hours. After the completionof the reaction, the reaction mixture was subjected to high-speedcentrifugation at about 10,000 G. This purification by centrifugationwas repeated until the electroconductivity of the separated supernatantfell below 20 μS/cm to remove the soluble components. Then, 10 liters ofethanol was added to the resultant dispersion while stirring the lattersufficiently. The white precipitate which consequently occurred wascollected by filtration under suction pressure. It was dried at 30° C.for 10 hours to obtain 5.2 g (yield: 52%) of carboxylmethylated starch,which is specific to an endo-type enzymatic reaction.

EXAMPLE 2

[0167] A reagent solution containing a cross-linking reagent was coatedon a colorless and transparent polyethylene terephthalate (PET) sheet(support) coated with a gelatin undercoating and having a thickness of180 μm. The sheet was then dried, forming a reagent layer wherein therespective components had the coverages as set forth below.Alkaline-treated Gelatin 14.5 g/m² Nonylphenoxypolyethoxyethanol 0.2g/m² (Containint 9 to 10 (average) of Oxyethylene Unites) Glucoseoxidase 5,000 U/m² Peroxidase 15,000 U/m² Glucoamylase 5,000 U/m²2-(4-hydroxy-3,5-dimethoxyphenyl)-4- 0.38 g/m²[4-(dimethylamino)phenyl]-5-phenethyl- imidazole (Leuco Dye) AcetateBis[(vinylsulfonylmethylcarbonyl)amino]-methane 0.1 g/m² An adhesivelayer was coated on the reagent layer to have the following coverage,and then dried. Alkaline-treated Gelatin 14.5 g/m²Bis[(vinylsulfonylmethylcarbonyl)amino]-methane 0.1 g/m²

[0168] Then, an aqueous solution containing the following reagent wascoated over the surface of the adhesive layer to have the followingcoverages to swell the gelatin layer and a tricot knitted cloth made byknitting PET spun yarn of 36 gage corresponding to 50 deniers and havinga thickness of about 250 μm was then laminated thereon, by pressing witha uniform light pressure to form a porous spreading layer.Nonylphenoxypolyethoxyethanol 0.15 g/m² (containing 9 to 10 (average) ofoxyethylene Units) Bis[(vinylsulfonylmethylcarbonyl)amino]-methane  0.4g/m²

[0169] Thereafter, a substrate layer was formed by coatingcarboxylmethylated starch specific to an endo-type enzymatic reactionprepared by Example 1, followed by drying, to have the followingcoverages, to prepare a multi-layered analysis element for thequantitative analysis of CRP. Carboxylmethylated starch 3.5 g/m²Mannitol 3.0 g/m² MES (2-morpholinoethane sulfonic acid) 2.0 g/m²

[0170] The thus preparedelement was cut into rectangular chips, 14 mm×13mm. The chips were severally encased with slide frames described inJP-A-57-63,452 to prepare multi-layered dry slide 1 for the analysis ofCRP according to the present example.

[0171] As a comparative example, a control slide 2 having the sameconstruction as the slides 1 of the example except that an untreatedcarboxylmethylated starch (Exprotab) was used as a substrate in theplace of the carboxylmethylated starch specific to the endo-typeenzymatic reaction.

EXAMPLE 3

[0172] The 50 mM glycerophosphate buffer solution (pH 7) was spotted ina unit amount of 10 μL severally on the dry slides 1 of Example 2 andthe dry slides 2 of Comparative Example. Thereafter, the slides werekept at 37° C. and measured from the PET supporting member side forreflected optical density at a central wavelength of 650 nm along thecourse of time. The time-course changes of the reflected optical densityafter the buffer-spotting in the are shown in FIG. 4.

[0173] On the control slides 2 of Comparative Example which useduntreated carboxylmethylated starch as a non-diffusible substrate, thereflected optical density was found to begin increasing along the courseof time after the spotting of the buffer solution (indicated by the mark-◯- in the diagram). This fact indicates that the untreatedcarboxylmethylated starch was decomposed into glucose by theglucoamylase (fragmenting enzyme), an exo-active enzyme, migrated fromthe reagent layer.

[0174] In contrast, on the slides 1 of Example 2 which usedcarboxylmethylated starch rendered specific to an endo-type enzymaticreaction in advance by the treatment with glucoamylase, the reflectedoptical density was constant and showed no increase (as indicated by themark -- in the diagram).

EXAMPLE 4

[0175] 10 μl for each of the 50 mM glycerophosphate buffer solution (pH7) containing an amylase-labelled anti-CRP-IgG (0.1 mg/mL) and a knownamount of CRP was spotted on the dry slides 1 of Example 2 and the dryslides 2 of Comparative Example. The slides 1 and 2 were maintained at37° C. and measured from the PET support side for reflected opticaldensity with a visible light having a central wavelength of 650 nm. Thedifferences in reflected optical density (ΔOD⁶⁻⁴) between 4 minutes and6 minutes after the spotting are shown in FIG. 5. It is clear from thecalibration curve of FIG. 5 that the dry immunoassay element of thisinvention using a carboxylmethylated starch rendered specific in advanceto the endo-type enzymatic reaction was capable of determining theamount of CRP with high accuracy (as indicated by the mark -- in thediagram). The changes of ΔOD₆₋₄ relative to the change of CRPconcentration were large as compared with those of the ComparativeExample (as indicated by the mark -◯- in the diagram), indicating animprovement in sensitivity.

EXAMPLE 5

[0176] A multi-layered analysis element having the same construction asthe element of Example 2 was prepared by faithfully repeating theprocedure of Example 2. On the tricot knitted cloth layer, which servedboth as a substrate layer and a spreading layer, an ethanol solution ofthe amylase-labelled CRP-IgG was coated and impregnated to have acoverage of 3 mg/m², followed by drying, to prepare a multi-layeredimmunoassay slide 3 for analysis of CRP (Example 5). As a ComparativeExample, the tricot knitted cloth layer of the multi-layered assayelement of the comparative slide 2 prepared in Example 2 was similarlycoated and impregnated with an ethanol solution of the amylase-labelledCRP-IgG at a coverage of 3 mg/m², and then dried to prepare acomparative multi-layered immunoassay slide 4.

EXAMPLE 6

[0177] On the slides 3 and slides 4, 10 μl for each of 50 mMglycerophosphate buffer solution (pH 7) containing CRP in a varyingconcentration was spotted. The slides were maintained at 37° C. andmeasured from the support side for reflected optical density at awavelength of 650 nm to find differences in reflected optical density(ΔOD⁶⁻⁴) between 4 minutes and 6 minutes after the spotting of thesolution. It is clear from the calibration curve of FIG. 6 that the dryimmunoassay element of this invention using the carboxylmethylatedstarch specific to the endo-type reaction (as indicated by the mark --in FIG. 6) was capable of performing quantitative assay of CRP with highsensitivity as compared with the comparative example (as indicated bythe mark -◯-).

EXAMPLE 7

[0178] The slides 3 of Example 5 and the slides 4 of the comparativeexample were studied comparatively for reproducibility (CV). On theslides 3 and slides 4, four samples (L-1, L-2, M, and H) of aCRP-containing human standard blood serum were spotted in a unit amountof 10 μL. The slides were maintained at 37° C. and measured from thesupporting member side for reflected optical density at a wavelength of650 nm to find differences in reflected optical density (ΔOD₆₋₄) between4 minutes and 6 minutes after the spotting of the solution. Acalibration curve for the slides 3 of Example 5 and a calibration curvefor the slides 4 of comparative example were separately prepared inadvance. From the values of ΔOD₆₋₄ of the samples consequently found,the relevant CRP concentration (measured values) were calculated. Thismeasurement was performed on each of the samples up to 10 repetitions.The averages, standard deviations (S.D.), and coefficient of variation(CV=(S.D./Aberage)×100) of the results of measurement were consequentlyobtained. The results are shown in Tables 1 and 2 given below. TABLE 1Reproducibility of Slides 3 (Example 5) Sample L-1 L-2 M H Measured 2.31.7 5.3 9.0 Value 2.6 1.6 5.4 8.7 (mg/dL) 2.4 1.4 5.3 9.0 2.4 1.5 5.19.4 2.2 1.5 5.0 9.2 2.1 1.5 5.3 8.7 2.4 1.6 5.3 8.9 2.3 1.5 5.9 9.2 2.21.5 5.6 9.1 2.2 1.6 5.5 9.5 Average 2.3 1.5 5.4 9.1 S.D. 0.1 0.1 0.3 0.3CV 6.4 5.3 4.9 3.0

[0179] TABLE 2 Reproducibility of Slides 4 (Comparative Example) SampleL-1 L-2 M H Measured 2.5 1.1 5.8 10.6 Value 2.7 1.9 6.1 10.4 (mg/dL) 2.01.9 6.2 9.7 2.2 1.9 5.6 10.3 2.1 1.9 5.3 10.4 2.9 2.2 5.9 12.1 3.3 2.35.4 12.9 3.1 2.1 5.8 11.2 2.1 2.3 5.8 12.4 2.3 1.9 5.9 13.6 Average 2.51.9 5.8 11.4 S.D. 0.5 0.3 0.3 1.3 CV 18.9 17.4 4.8 11.5

[0180] It is noted from Tables 1 and 2 that the slides 3 of Example 5 ofthis invention using the substrates specific to the endo-type reactionshowed only slight variation and excelled in reproducibility, whereasthe conventional slides 4 for comparison showed a wide variation ofmeasured value (concentration). Particularly, in the low concentrationrange (samples L-1 and L-2) and the high concentration range (sample H),the CV values of the slides 3 of working example were about ⅓ lower thanthose of the slides 4 for comparison, indicating that the immunoassayelements according to the present invention excelled in reproducibilityand in accuracy as well.

EXAMPLE 8

[0181] The slides 3 of Example 5 and the slides 4 for comparison werestudied comparatively for stability of preservation. Since the dry assayelements are generally stable for a duration of about one month. In thepresent example, therefore, the slides were preserved at 25° C. by wayof acceleration of test and the values of measurement obtained one day,three days, and seven days after manufacture of slide were compared withthe values of measurement obtained immediately (0 day) after manufactureof slide. On the slides 3 (Example 5) and the slides 4 (comparison)after specified numbers of days following manufacture of slide, aCRP-containing standard samples CP1 (1.4 mg/dL), CP2 (4.2 mg/dL), andCP3 (10.0 mg/dL) were severally spotted in a unit amount of 10 μL. Theseslides were maintained at 37° C. and measured from the supporting memberside for reflected optical density at a wavelength of 650 nm to find thedifferences in reflected optical density (ΔOD₆₋₄) between 4 minutes and6 minutes after the deposition of the solution in the form of a spot.The results are shown in Tables 3 and 4 below. TABLE 3 Storage Stabilityof Slides 3 (Example 5) ΔOD⁶⁻⁴ after storage of Slide 0 day 1 day 3 days7 days CP1 0.3432 (100%) 0.3355 (98%) 0.3339 (97%) 0.3276 (96%) CP20.2734 (100%) 0.2685 (98%) 0.2610 (96%) 0.2658 (97%) CP3 0.2045 (100%)0.2065 (101%) 0.2002 (98%) 0.1938 (95%)

[0182] TABLE 4 Storage Stability of Slides 4 (Comparative Example)ΔOD⁶⁻⁴ after storage of Slide 0 day 1 day 3 days 7 days CP1 0.3358(100%) 0.3271 (97%) 0.3171 (94%) 0.2884 (86%) CP2 0.2536 (100%) 0.2467(97%) 0.2285 (90%) 0.2138 (84%) CP3 0.1753 (100%) 0.1656 (94%) 0.1587(91%) 0.1390 (79%)

[0183] On the slides 4 (for comparison) using the conventional untreatedcarboxylmethylated starch as the substrate, the values of ΔOD₆₋₄decreased with passage of days of preservation at 25° C., decreases of5-10% found on the third day and large decreases in the approximaterange of 15-20% on the seventh day. In contrast thereto, on the slides 3(Example 5) using the present invention's carboxylmethylated starchprepared as a substrate specific to the endo-type reaction, thedecreases of the value of ΔOD₆₋₄ were only 2-4% on the third day andabout 3-5% on the seventh day respectively of preservation. The resultsindicate that the immunoassay elements according to the presentinvention excelled in storage stability.

EXAMPLE 9

[0184] Preparation of Endo-Type Selectively Reactive Substrate;

[0185] In 8,960 g of super-pure water, 453 g of carboxylmethylatedstarch (made by Edward Mendel Company Inc. and sold under a trademarkdesignation of “Exprotab”) was stirred for about 4 hours and swelled.The swelled starch was alkalinized by addition of 1139 g of 0.5N NaOHand stirring for about one hour. Thereafter, it was reverted to pH 7 bythe addition of 32 g of acetic acid. It was adjusted to pH 5.7 with anMES (2-morpholinoethane sulfonate) buffer solution and then caused toreact with 80 g of glucoamylase enzyme solution (320 k Unit/L, 5 mM MES,pH 6.0, made by Toyobo K.K.) at 37° C. for 12-20 hours. The reactionsolution consequently obtained was diluted with pure water to a totalvolume of 140 L. The resultant suspension was passed through a ceramicmembrane filter (monolithic type, 2 μm in pore size and 0.24 m² inmembrane area, made by Nippon Gaishi K.K. and sold under a registeredtrademark of “CEFILT-MF”). The ceramic membrane filter was deprived of asoluble component by circulating pure water through the filter until theelectroconductivity of the filtered water fell below 20 μS/cm. Theresidue of the carboxylmethylated starch was collected from the filterand diluted with purified water to a total volume of 100 L. About 3.6 Lof the diluted residue was centrifuged at 7,500 rpm for 10 minutes andthe residue of centrifugation was collected. About 18 L of isopropylalcohol was added to the residue, and the white substance consequentlyprecipitated was collected by filtration under reduced pressure. Theprocedure from the centrifugation through the precipitation of isopropylalcohol was performed up to about 26 repetitions and the residuesconsequently formed in the successive rounds of procedure were gatheredand dried at 35° C. for 24 hours. The operation afforded 220 g ofglucoamylase-treated carboxylmethylated starch specific to an endo-typeenzymatic reaction (hereinafter referred to as “GA-treated CMS”).

[0186] As a comparative example (Control), a carboxylmethylated starchwas prepared by faithfully repeating the operation mentioned above whileomitting the treatment with glucoamylase (non-glucoamylase-treatedcarboxylmethylated starch; hereinafter referred to as “untreated CMS”).

EXAMPLE 10

[0187] The GA-treated CMS and the untreated CMS obtained in Example 9were tested for degree of carboxylmethylation and the dispersionsthereof in water were tested for degree of swelling. The degree ofcarboxylmethylation was determined by finding the ratio of glucose unitmodified with a carboxylmethyl group to the whole glucose unit by meansof ¹³C-NMR. The degree of swelling was determined by preparing 10 mL ofan aqueous 0.7% solution of a given CMS, placing the aqueous solution ina test tube, allowing it to stand at rest at 25° C. for 30 minutes, andthen measuring the volume (mL) of CMS consequently settled to the bottomof the test tube. The volume was reported as the degree of swelling.

[0188] It is noted from the results given in Table 5 below that thecarboxylmethylated starch treated with the glucoamylase gained in degreeof carboxylmethylation and that 28% of the whole glucose units formedtherein possessed a carboxylmethyl group. This fact indicates that thetreatment with glucoamylase resulted in decreasing the amount ofnon-CM-modified glucose units in the CM-modified starch and consequentlyincreasing the ratio of the CM-modified glucose units because thetreatment hydrolyzed the glucoside bonds from the non-reducing terminalthrough the point immediately preceding the point ofcarboxylmethylation. It is also conceivable that the increase in theratio of incorporation of the carboxylmethyl group, a hydrophilic group,resulted in adding to the ease of hydration and exalting the degree ofswelling. TABLE 5 Untreated CMS GA-treated CMS Degree of 21% 28%Carboxylmethylation Degree of swelling  4.5  5.5

EXAMPLE 11

[0189] Reactivity of GM-Treated CMS with Glucoamylase;

[0190] The glucoamylase-treated carboxylmethylated starch obtained inExample 9 and the non-glucoamylase-treated carboxylmethylated starch ofcomparative example were severally dispersed in a 5 mM MES buffersolution (pH 6.0, containing 0.5% of Block Ace (made by Snow Brand MilkProducts Co., Ltd.) and 68 μM of CaCl₂) to produce 0.2% (W/V)dispersions. To 7 mL of the 0.2% CMS dispersions, 70 μL of glucoamylasesolution (made by Toyobo K.K., 320 k Unit/L, 5 mM MES, pH 6.0) wasadded, and shaken together at 37° C. for 60 minutes at 120 rpm to inducea reaction. The reaction solutions consequently obtained were combinedwith 70 μL of 10% phosphoric acid and ice cooled to stop the reaction.The resultant reaction solutions were centrifuged at 12,000 rpm for 4minutes to separate the unaltered CMS. One hundred (100) μL of thesupernatant of centrifugation containing the produced glucose was addedto 3 mL of a reaction solution A of the following composition and theywere together shaken at 37° C. for 30 minutes at 120 rpm to induce acoloring reaction. The resultant reaction solutions were tested forabsorbancy at a wavelength of 727 nm. Relevant calibration curves wereprepared by adding 100 μL of a glucose solution of known concentrationto 3 mL of the reaction solution A and subjecting the resultant mixturesto the same coloring reaction.

[0191] Reaction Solution A Leuco dye 3.9 mg(N-(carboxymethylaminocarbonyl)-4,4′- bis(dimethylamino)-diphenylaminesodium salt) (Wako Junyaku K.K. product code; “DA-64”) Glucose oxidase139 units (Toyobo K.K: product code; “GLO-501”) Peroxidase 115 units(Toyobo K.K. product code; “PEO-301”) 100 mM MES buffer solution (pH6.0) 100 mL

[0192] The measurement was performed on the GA-treated CMS up to fiverepetitions and on the untreated CMS up to two repetitions. It is notedfrom the results given in Table 6 that the CM starch not treated withglucoamylase produced about 33 μg of glucose per gram, whereas the CMstarch treated in advance with glucoamylase only produced an average ofnot more than 0.1 μg of glucose. This fact indicates that nearly all thenon-reducing terminal glucose of the GA-treated CMS of this inventionwas either modified or positioned at the point of chain branching andwould not form a substrate of glucoamylase, an exo-reactive type enzyme.TABLE 6 Glucose produced from 1 g of CMS Average GA-treated CMS   0.25(μg)   0.07 −0.02   0.08   0.08  0.09 (μg) Untreated CMS 33.56 (μg)33.63 33.60 (μg)

EXAMPLE 12

[0193] Reactivity of GA-Treated CMS with α-Amylase (Wet System)

[0194] The glucoamylase-treated carboxylmethylated starch obtained inExample 9 and the non-glucoamylase-treated carboxylmethylated starch ofcomparative example were severally dispersed in a 5 mM MES buffersolution (pH 6.0, containing 0.5% of Block Ace (made by Snow Brand MilkProducts Co., Ltd.) and 68 μM of CaCl₂) to produce 0.2% (W/V)dispersions. The 0.2% CMS dispersions, 7 mL in volume, and 70 μL of anα-amylase solution of a varying concentration added severally theretowere shaken together at 37° C. for 60 minutes at 120 rpm to induce areaction. The reaction solutions obtained consequently were combinedwith 70 μL of 10% phosphoric acid and ice cooled to stop the reaction.The resultant reaction solutions were centrifuged at 12,000 rpm for 4minutes to effect separation of the unaltered CMS. One hundred (100) μLof the supernatant of centrifugation containing the produced glucose wasadded to 3 mL of a reaction solution B of the following composition andthey were together shaken at 37° C. for 30 minutes at 120 rpm to inducea coloring reaction. The resultant reaction solutions were tested forabsorbancy at a wavelength of 727 nm. Relevant calibration curves wereprepared by adding 100 μL of a glucose solution of known concentrationto 3 mL of the reaction solution B and subjecting the resultant mixturesto the same coloring reaction. A graph was obtained by plotting thecontents of oligosaccharides formed based on the calibration curves(FIG. 7).

[0195] Reaction Solution B Leuco dye 3.9 mg(N-(carboxymethylaminocarbonyl)-4,4′- bis(dimethylamino)-diphenylaminesodium salt) (Wako Junyaku K.K; product code: “DA-64”) 3.9 mgGlucoamylase 180 units (Toyobo K.K. product code: “GLA-111”) Glucoseoxidase 139 units (Toyobo K.K; product code: “GLO-501”) Peroxidase 115units (Toyobo K.K; product code: “PEI-301”) 100 mM MES buffer solution(pH 6.0) 100 mL

[0196] As shown in FIG. 7, the CM-starch treated in advance withglucoamylase had the reactivity to the α-amylase (an endo-reaction typeenzyme) (the inclination of the calibration curve in the diagram), wasabout one half of that of the CM-starch not treated in advance. Whilethe background value of the untreated CMS was about 1.3 ppm, that of theGA-treated CMS was nearly zero.

[0197] The fact that the content of oligosaccharide produced from theuntreated CMS was not zero while the content of α-amylase was zero maybe explained by the following supposition. The vertical axis of the dataof FIG. 7 is the scale of oligosaccharide formed, however, in thereaction system of the present example, the amount of the ultimatelyproduced glucose was analyzed. Although the residues of CMS (CM-starch)remaining after the reaction with the α-amylase were removed bycentrifugation, minute amounts of CMS particles survived in thesupernatants. When the minute amounts of CMS particles are suffered tomingle into the supernatants collected as the produced oligosaccharides,the CMS particles are also destined to be attacked by the glucoamylasecontained in the coloration reaction solution B. In the untreated CMS,since the terminal glucose was not removed, the terminal glucose washydrolyzed by the glucoamylase and consequently detected. Since theamount of the terminal glucose thus detected did not dependent on theamount of α-amylase used but was nearly constant, it was fated to raisethe whole calibration curve as the background value in FIG. 7.

[0198] In contrast, the CM-starch treated in advance with theglucoamylase was devoid of an unmodified glucose terminal capable ofreacting with glucoamylase which is an exo-active type enzyme. Even whenthe CMS particles are suffered to mingle into the coloration reactionsystem, they will not raise the background value.

[0199] The fact that the reactivity of the CM-starch with the α-amylaseis lowered by the treatment with glucoamylase may be logically explainedby the following supposition. The α-amylase exhibits reactivity topolysaccharides having not less than four molecules of glucose chainlength. This reactivity increases in accordance as the degree ofpolymerization increases (the chain length gains in size). When theglucose unit in the sugar chain is carboxylmethylated at random, thepart formed of continued glucose molecules to which the α-amylaseexhibits high reactivity is inevitably decreased. As a result, thestarch inherently has the reactivity thereof to the α-amylase loweredwhen it is carboxylmethylated. When the CM-starch is further treatedwith glucoamylase, the branched chains of the CM-starch are severallyremoved through decomposition from the non-reducing terminal. When CMsites are present halfway along the lengths of branched chains, they arehydrolyzed and removed by glucose units from the non-reducing terminalup to the sites. As a result, the CM-starch treated with theglucoamylase are converted into a kind of limit dextrins and the partformed of continued glucose molecules to which the α-amylase exhibitshigh reactivity is further decreased apparently. FIG. 7 demonstratesthis fact.

[0200] Though the CM-starch treated in advance with glucoamylase has thereactivity with the α-amylase lowered as described above, it has thereactivity with the α-amylase increased in the dry assay element asdemonstrated in Examples 13 and 14 to be cited herein below.

EXAMPLE 13

[0201] Reactivity of GA-Treated CMS with α-Amylase (Dry System)

[0202] A multi-layered analysis element identical in construction withthat of Example 5 was prepared by faithfully repeating the procedure ofExample 5 while using the glucoamylase-treated CM-starch obtained inExample 9 instead.

[0203] Specifically, slides identical in construction to those ofExample 2 were prepared by faithfully repeating the procedure of Example2 while using the glucoamylase-treated CM-starch obtained in Example 9as the substrate layer in the place of the carboxylmethylated starchused in Example 2. Thereafter, in the same manner as in Example 5, onthe tricot knitted cloth layer, which served both as a substrate layerand a spreading layer, an ethanol solution of the amylase-labelledCRP-IgG was coated and impregnated at a coverage of 3 mg/m², and thendried to prepare multi-layered immunoassay slide 5 for analysis of CRP(Example 13).

[0204] As a comparative example, a slide 6 example identical inconstruction to that of Example 13 was prepared by repeating theprocedure of Example 13 while using an untreated carboxylmethylatedstarch as the substrate in the place of the glucoamylase-treatedcarboxylmethylated starch.

EXAMPLE 14

[0205] On the slide 5 and the slide 6, 10 μL for each of 50 mMglycerophosphorate buffer solution (pH 7) containing CRP in a varyingconcentration of 1, 1.4, 4.2, and 10 mg/dL was spotted. The slides weremaintained at 37° C. and were measured from the support side along thecourse of time for reflected optical density at a wavelength of 625 nm.The time-course changes of reflected optical density after the spottingof the CRP solution are shown in FIG. 8.

[0206] When the CRP solution of 0 mg/dL isspotted, the hydrolyticactivity with the CM-starch, a non-diffusible substrate, is maximizedbecause the α-amylase in the labelledantibody is not subjected to thesteric hindrance due to the binding with an antigen (CRP). The activityexistent at the CRP concentration of 0 mg/dL at which the α-amylaseexhibits the highest activity was notably high on the slide 5 (Example14, the righthand side of FIG. 8) using the GA-treated CMS as comparedwith the comparative example (slide 6 using untreated CMS, on thelefthand side of FIG. 8). Based on the index of the slope of thetime-course change of reflected optical density, the CM-starch treatedin advance with glucoamylase showed about twice as high reactivity withthe α-amylase as the untreated CM-starch.

[0207] When the steric hindrance to the α-amylase gained in intensity inaccordance as the content of CRP, an antigen, the decline of thereactivity with the α-amylase was conspicuous on the slide 5 (Example14, on the righthand side of FIG. 8) using the GA-treated CMS. This factindicates that owing to the reactivity of the α-amylase with theCM-starch in the dry assay element, the change of the reflected opticaldensity relative to the change of CRP concentration is increased and thesensitivity is enhanced by treating the starch in advance withglucoamylase and consequently rendering it specific to the endo-activetype enzymatic reaction.

What is claimed is:
 1. An immunoassay element for quantitativelyanalyzing an antigen by determining the change in enzymatic activitycaused by any of 1) a reaction between the antigen and anenzyme-labelled antibody; 2) a reaction between the antigen, an antibodyand an enzyme-labelled antigen; and 3) a reaction between the antigen,an enzyme-labelled antibody and a conjugate of the antigen with a highmolecular weight compound; wherein said element comprises a substratelayer containing a non-diffusible substrate which forms a diffusiblematerial in the presence of said labelling enzyme, and a reagent layercontaining a fragmenting enzyme for further fragmenting said diffusiblematerial into a lower molecular weight product: characterized in thatsaid non-diffusible substrate is a substrate which reacts solely withsaid labelling enzyme and avoids reacting with said fragmenting enzyme.2. The immunoassay element according to claim 1, wherein saidnon-diffusible substrate is a macromolecular polysaccharide, saidlabelling enzyme is an endo-active glucosidase, and said fragmentingenzyme is an exo-active glucosidase.
 3. The immunoassay elementaccording to claim 1, wherein said labelling enzyme is an endo-activeglucosidase, said fragmenting enzyme is an exo-active glucosidase, andsaid non-diffusible substrate is an endo-type selectively reactivesubstrate having a modified non-reducing terminal glucose.
 4. Theimmunoassay element according to claim 3, wherein said endo-typeselectively reactive substrate has the non-reducing terminal glucosethereof modified with a carboxylmethyl group.
 5. The immunoassay elementaccording to claim 3, wherein said labelling enzyme is α-amylase, saidfragmenting enzyme is glucoamylase or α-glucosidase, and saidnon-diffusible substrate is carboxylmethylated starch restrictivelydecomposed in advance with an endo-active glucosidase from thenon-reducing terminal glucose site through the carboxylmethyl-modifiedglucose unit site or the glucose chain branching site.
 6. Theimmunoassay element according to claim 1, wherein said reagent layer isformed of a layer containing a hydrophilic polymer as the binder.
 7. Theimmunoassay element according to claim 1, wherein said substrate layeris a porous layer formed of a porous medium.
 8. The immunoassay elementaccording to claim 1, wherein said enzyme-labelled antibody is containedin said substrate layer or a layer superposed on said substrate layer.9. The immunoassay element according to claim 1, wherein said antibodyis contained in said substrate layer or a layer superposed on saidsubstrate layer.
 10. The immunoassay element according to claim 1,wherein said enzyme-labelled antigen is contained in said substratelayer or a layer superposed on said substrate layer.
 11. The immunoassayelement according to claim 1, wherein said antibody and saidenzyme-labelled antigen are contained in said substrate layer or a layersuperposed on said substrate layer.
 12. The immunoassay elementaccording to claim 1, wherein said conjugate of the antigen with thehigh molecular weight compound and said enzyme-labelled antibody arecontained in said substrate layer or a layer superposed on saidsubstrate layer.
 13. The immunoassay element according to claim 1,further comprising a reagent composition for reacting with said lowermolecular weight product to form a dye having an absorption peak in thevisible wavelength range and contained in said reagent layer or anotherwater-permeable layer.
 14. The immunoassay element according to claim13, wherein the reagent composition contains a leuco dye which colorsupon oxidation.
 15. The immunoassay element according to claim 14,wherein said reagent layer contains a hydrophilic binder, and thereagent composition contains a dispersion of a solution of leuco dye ina water-insoluble solvent in the hydrophilic binder.
 16. The immunoassayelement according to claim 15, wherein said reagent composition containsa peroxidase and the leuco dye.
 17. An immunoassay element forquantitatively analyzing an antibody by determining the change inenzymatic activity caused by a reaction between the antibody and anenzyme-labelled antigen or a reaction between the antibody, an antigenand an enzyme-labelled antibody, wherein said element comprises asubstrate layer containing a non-diffusible substrate which forms adiffusible material in the presence of the labelling enzyme, and areagent layer containing a fragmenting enzyme for further fragmentingsaid diffusible material into a lower molecular weight product:characterized in that said non-diffusible substrate is a substrate whichreacts solely with said labelling enzyme and avoids reacting with saidfragmenting enzyme.
 18. The immunoassay element according to claim 17,wherein said non-diffusible substrate is a macromolecularpolysaccharide, said labelling enzyme is an endo-active glucosidase, andsaid fragmenting enzyme is an exo-active glucosidase.
 19. Theimmunoassay element according to claim 17, wherein said labelling enzymeis an endo-active glucosidase, said fragmenting enzyme is an exo-activeglucosidase, and said non-diffusible substrate is an endo-typeselectively reactive substrate having a modified non-reducing terminalglucose.
 20. The immunoassay element according to claim 19, wherein saidendo-type selectively reactive substrate has the non-reducing terminalglucose thereof modified with a carboxylmethyl group.
 21. Theimmunoassay element according to claim 19, wherein said labelling enzymeis α-amylase, said fragmenting enzyme is glucoamylase or α-glucosidase,and said non-diffusible substrate is carboxylmethylated starchrestrictively decomposed in advance with an endo-active glucosidase fromthe non-reducing terminal glucose site through thecarboxylmethyl-modified glucose unit site or the glucose chain branchingsite.
 22. The immunoassay element according to claim 17, wherein saidreagent layer is formed of a layer containing a hydrophilic polymer asthe binder.
 23. The immunoassay element according to claim 17, whereinsaid substrate layer is a porous layer formed of a porous medium. 24.The immunoassay element according to claim 17, wherein saidenzyme-labelled antibody is contained in said substrate layer or a layersuperposed on said substrate layer.
 25. The immunoassay elementaccording to claim 17, wherein said antibody is contained in saidsubstrate layer or a layer superposed on said substrate layer.
 26. Theimmunoassay element according to claim 17, wherein said enzyme markerantigen is contained in said substrate layer or a layer superposed onsaid substrate layer.
 27. The immunoassay element according to claim 17,wherein said antibody and said enzyme-labelled antigen are contained insaid substrate layer or a layer superposed on said substrate layer. 28.The immunoassay element according to claim 17, further comprising areagent composition for reacting with said lower molecular weightproduct to form a dye having an absorption peak in the visiblewavelength range and contained in said reagent layer or anotherwater-permeable layer.
 29. The immunoassay element according to claim28, wherein the reagent composition contains a leuco dye which colorsupon oxidation.
 30. The immunoassay element according to claim 29,wherein said reagent layer contains a hydrophilic binder, and thereagent composition contains a dispersion of a solution of leuco dye ina water-insoluble solvent in the hydrophilic binder.
 31. The immunoassayelement according to claim 30, wherein said reagent composition containsa peroxidase and the leuco dye.